Enzyme-linked immunosorbent assay antigen quantitation

Engvall E, Jonsson K, Perlmann P. 1971. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta 251 427-434. 

Engvall, E. and Perlman, P. (1972) Enzyme-linked immunosorbant assay, ELISA. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109,129. 

Yl. Yamagata, H., Harley, J. B., and Reichlin, M., Molecular properties of the Ro/SS-A antigen and enzyme-linked immunosorbent assay for quantitation of antibody. J. Clin. Invest. 74, 625—633... 

Edwards JH (1972) The isolation of antigens associated with farmer s lung. Clin Exp Immunol 11 341-355 Engwall E and Perlman P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8 871-874 Erkinjuntti-Pekkanen R, Reiman M, Kokkarinen JI et al (1999) IgG antibodies, chronic bronchitis, and pulmonary function values in farmer s lung patients and matched controls. Allergy 54 1181-1187... 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Antibodies can be combined with enzymes and color reagents or radioactive antigens to produce quantitative testing for drugs. The ELISA or Enzyme-Linked ImmunoSorbant Assay uses antibodies generated against the Ag to be tested for covalently linked to an enzyme which can catalyze a color change reaction such as the NADH to NAD conversion (Xmax at 340 nm). When the Ag-Ab complex is formed the enzyme is activated and the color can be detected. 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

Quinn, C.P., Semenova, V.A., Elie, C.M., Romero-Steiner, S., Greene, C., Li, H., Stamey, K. (2002). Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen. Emerg. Infect. Dis. 8 1103-10. 

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for maduramicin in poultry feed. The assay utilized polyclonal anti-maduramicin antibody raised in rabbits, maduramicin monoamide with 1,6-hexane diamine-conjugated ovalbumin as the coating antigen, horseradish peroxidase conjugated goat anti-rabbit IgG and 2,2 azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS) for quantitation. Standard curves ranging from 0 to 80 ng/mL maduramicin were constructed. The assay did not cross-react with monensin, lasalocid, salinomycin, lincomycin, narasin, chlortetracycline or roxarsone. Broiler feed fortified at 4 to 7 ppm maduramicin were shown to be quantifiable by ELISA at an average recovery of 98.1%. This ELISA method for maduramicin in poultry feed is comparable to the established HPLC-F method. 

Enzyme-Linked Immunosorbent Assay. LlSAis a heterogeneous EIA technique that is widely used in clinical analyses. In this type of assay, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as that of a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates separation of bound- and free-labeled reactants. In the most common approach to using the ELISA technique, an aliquot of sample or calibrator containing the antigen to be quantitated is added to and allowed to bind with a solid phase antibody. After washing, enzyme-labeled antibody is... 

After more than two decades, advances in IHC have provided a feasible approach to performing immunostain-ing on routinely processed tissues, such that this method is now routine for the performance of IHC special stains in surgical pathology laboratories using EEPE tissues (see Appendix lA). However, demands for improved reproducibility and for quantification have led to a growing recognition that IHC has the potential to be more than just a special stain. If properly controlled in all aspects of its performance, IHC can provide a tissue-based immunoassay with the reproducibility and quantitative characteristics of an ELISA (enzyme-linked immunosorbent assay) test, which not only detects the presence of an analyte (protein or antigen) but also provides an accurate and reliable measure of its relative or real amount (see Quality Control and Standardization section). 

Cell-enzyme-linked immunosorbent assay (cell-ELISA) is an useful technique for the quantitative analysis of cell surface antigen expression that was developed on the basis of enzyme immunohistochemistry (EIH) and ELISA. Since its development, which was made possible by the establishment of monoclonal antibody technology, a wide range of cell types and surface molecules were analyzed by cell-ELISA. Here we show four variants of this method and provide a brief comparison of cell-ELISA with flow cytometry (FACS) and radioimmunobinding assay (BIA), which are other methods for the quantitative detection of cell-surface molecules. We describe step-by-step procedures for both direct and indirect cell-ELISA using either adherent or nonadherent live cells. 

The enzyme-linked immunosorbent assay (ELISA or EIA) is one of the most commonly utilized methods used in protein detection and analysis. An ELISA can provide quantitative informatimi about antigen or antibody concentrations in... 

The enzyme-linked immunosorbent assay (ELISA or EIA) is one of the most commonly utilized methods used in protein detection and analysis. An ELISA can provide quantitative information about antigen or antibody concentrations in solution by comparing the results of an unknown sample assay to a calibration curve based on known standard concentrations of the antibody or antigen of interest. Although, there are many variations in how ELISA may be performed, three of the most commonly used representative methods are discussed here (Fig. 1). The choice of which ELISA technique is used often depends on the nature of the antigen or antibody of interest, the availability of appropriate binding pairs, and the specificity of only the antigen of interest to a monoclonal antibody. 

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